Autoantibodies against the amino terminal of alpha-enolase are a useful diagnostic marker of Hashimoto's encephalopathy.

We investigated autoantibodies and their epitope(s) in Hashimoto's encephalopathy associated with Hashimoto's thyroiditis. In a proteomic analysis, they proved to recognize alpha-enolase. We further searched the epitope region in alpha-enolase using different regions of recombinant proteins expressed in cultured human cells. The amino terminal region was recognized by autobodies from a much higher proportion of patients with Hashimoto's encephalopathy (83.3%; 5/6) than from patients with Hashimoto's thyroiditis (11.8%; 2/17), and not at all by sera from controls (25 healthy individuals and 25 controls with other neurological disorders) (0%; 0/50). Neither the carboxyl terminal nor the mid-region of alpha-enolase showed specificity for Hashimoto's encephalopathy. Autoantibodies against the amino terminal of alpha-enolase are a useful diagnostic marker for Hashimoto's encephalopathy.

Molecular epidemiology of Neisseria gonorrhoeae exhibiting decreased susceptibility and resistance to ciprofloxacin in Hawaii, 1991-1999.

BACKGROUND: Clinically significant resistance to Centers for Disease Control and Prevention (CDC)-recommended doses of fluoroquinolones (ciprofloxacin and ofloxacin) has been reported for Neisseria gonorrhoeae. In Hawaii, fluoroquinolone-resistant gonococcal isolates were first identified in 1991. GOAL: To assess the diversity, based on phenotypic and genotypic characterization, of gonococcal isolates exhibiting decreased susceptibility (CipI; MICs = 0.125-0.5 microg/ml) or clinically significant resistance (CipR; MICs > or = 1 microg/ml) to ciprofloxacin in Hawaii from 1991 through 1999. STUDY DESIGN: Antimicrobial susceptibilities, auxotype/serovar (A/S) class, GyrA/ParC alteration patterns, and plasmid profiles were determined for gonococci isolated in Honolulu from 1991 through 1999 that exhibited intermediate or clinically significant resistance to ciprofloxacin. Strain phenotypes were defined by A/S class, GyrA/ParC alteration pattern, and penicillin-tetracycline resistance phenotype supplemented with plasmid profiles for beta-lactamase-producing isolates. RESULTS: Altogether, 68 isolates exhibiting intermediate or clinically significant resistance to ciprofloxacin belonged to 23 and 19 strain phenotypes, respectively. Among the CipI and CipR isolates, 4 and 13 GyrA/ParC alterations patterns were identified, respectively. The 91,95/Asp-86 alteration pattern occurred most frequently among CipR isolates. Forty-four strain phenotypes were represented by only one isolate. In addition, seven pairs and two clusters of isolates were identified. CONCLUSIONS: From 1991 through 1997, few gonococcal strains exhibiting intermediate or clinically significant resistance to CDC-recommended doses of fluoroquinolones were identified from Hawaii. Isolates belonged to a large number of phenotypic and genotypic types, suggesting that most cases were imported, with only a few instances in which isolate pairs indicated that secondary transmission of infections had occurred in Hawaii. Beginning in 1998, the number of CipR isolates increased markedly, and more isolates belonged to fewer phenotypic and genotypic types, suggesting either more frequent importation of fewer strain types or the possibility that the endemic spread of a few strains is beginning to occur.

Evidence for widespread infection of wild rats with hepatitis E virus in the United States.

Hepatitis E is an important medical pathogen in many developing countries but is rarely reported from the United States, although antibody to hepatitis E virus (anti-HEV) is found in > 1% of U.S. citizens. Zoonotic spread of the virus is suspected. Sera obtained from 239 wild rats trapped in widely separated regions of the United States were tested for anti-HEV. Seventy-seven percent of rats from Maryland, 90% from Hawaii, and 44% from Louisiana were seropositive for anti-HEV. Rats from urban as well as rural areas were seropositive and the prevalence of anti-HEV IgG increased in parallel with the estimated age of the rats, leading to speculation that they might be involved in the puzzling high prevalence of anti-HEV among some U.S. city dwellers. The discovery of a in rats in the United States and the recently reported discovery that HEV is endemic in U.S. swine raise many questions about transmission, reservoirs, and strains of HEV in developed countries.

Multiple photorefractive keratectomy retreatments for myopia.

PURPOSE: To examine the outcome of multiple photorefractive keratectomy retreatments by the excimer laser on eyes treated for myopia and myopic astigmatism. METHODS: We present 980 of 1218 (80%) consecutive eyes treated with a VISX 20/20 excimer laser that were available for follow-up. Thirteen eyes of 13 individuals were retreated twice using the same VISX excimer laser because of refractive regression. Epithelium was mechanically removed in the initial treatment and subsequent retreatment of all the eyes. No increased difficulty was noted upon removing the epithelium on subsequent retreatments. The aim in both the initial and subsequent treatments was to correct 100% of the refractive error. RESULTS: Eyes with higher amounts of myopia and astigmatism were more likely to require multiple retreatments. Seven eyes (54%) attained a spectacle-corrected visual acuity of 20/20 or better. Twelve eyes (92%) attained a spectacle-corrected visual acuity of 20/40 or greater following the second retreatment. The amount of astigmatism corrected was improved in 10 of the 13 eyes with an average improvement of 1.90 D of cylinder. Only one patient experienced a haze score of 3 or more following retreatment. CONCLUSION: There is a subgroup of patients who require multiple PRK retreatments for myopic regression. A good visual outcome may be obtained with minimal haze.

A new type of hereditary motor and sensory neuropathy linked to chromosome 3.

We report the clinical, pathological, and genetic findings of 23 patients in 8 families with hereditary motor and sensory neuropathy (proximal dominant form) (HMSN-P) in Okinawa, Japan. The clinical features were unique with respect to autosomal dominant inheritance, Kennedy-Alter-Sung syndrome-like proximal dominant neurogenic atrophy, obvious sensory involvement, painful muscle cramp, fasciculations, areflexia, and high incidences of elevated creatine kinase levels, hyperlipidemia, and diabetes mellitus. Electrophysiological and pathological studies revealed typical motor and sensory axonal neuropathy, and decreased numbers of anterior born and dorsal ganglion cells, which suggested the presence of neuronopathy in HMSN-P. Genetic linkage studies showed a lod score of 4.04 (two-point analysis) in DNA marker D3S1284. Haplotype analysis showed that the gene locus of the disease was mapped to 3p14.1-q13 bracketed by D3S1285 and D3S1281. In this region, the patients' chromosomes showed an obvious increase in the allele frequency of five markers. One allele in D3S1591 was identical in all patients but had a low frequency in the control population. This finding suggested the presence of linkage disequilibrium and a common origin of this allele in all patients with HMSN-P. The HMSN-P described here is a new clinical entity characterized by unique clinical manifestations and a new gene locus.

Predictability of excimer laser treatment of myopia and astigmatism by the VISX Twenty-Twenty. Melbourne Excimer Laser Group.

BACKGROUND: To determine the predictability of excimer laser photorefractive keratectomy (PRK) to correct myopia, astigmatism, or both between -1.00 and -19.00 diopters (D). SETTING: Royal Victorian Eye and Ear Hospital, East Melbourne, Australia. METHODS: This study comprised 1218 consecutive eyes treated with a VISX Twenty-Twenty excimer laser and followed prospectively for 12 months. Low myopia was treated with one ablation zone (6.0 mm), high myopia with two ablation zones (5.0 and 6.0 mm), and extreme myopia with three ablation zones (4.5, 5.0, and 6.0 mm). Maximum spherical treatment was 15.00 D at the corneal plane. Data were analyzed to determine the predictability of the postoperative outcomes by preoperative refraction. RESULTS: Nine hundred eighty eyes (80.5%) were available for the 12 month follow-up. The predictability of refraction and uncorrected and best corrected visual acuities progressively decreased with increasing myopia, although a comparable percentage of spherical correction was achieved at each diopter of myopia. The likelihood of losing lines of best corrected visual acuity and corneal haze increased with increasing myopia. CONCLUSION: These data can be used to counsel patients of likely outcomes of excimer laser PRK to correct myopia.

Diversity of isolates of penicillinase-producing Neisseria gonorrhoeae (PPNG) in Honolulu, Hawaii: 1982-1991.

BACKGROUND AND OBJECTIVES: Gonococcal infections caused by penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates have increased in geographic distribution and prevalence. It was postulated that PPNG strains would become endemic in Honolulu and that, in turn, this city would serve as a reservoir for the introduction of PPNG strains into the continental United States. GOAL OF THIS STUDY: To assess the role of Honolulu as a reservoir for PPNG strains by assessing the diversity and persistence of PPNG strains between 1982 and 1991. STUDY DESIGN: A total of 432 PPNG strains were characterized by auxotype/serovar (A/S) class and plasmid content, and their distribution during the 10-year period was studied. RESULTS: Of 432 isolates, 373 (86.4%) possessed a 4.4-Mdal beta-lactamase plasmid; 39 (9.0%) possessed a 3.2-Mdal beta-lactamase plasmid; and 20 (4.6%) possessed a 3.05-Mdal beta-lactamase plasmid. A total of 53 A/S classes were identified. Asian, African, and Toronto PPNG strains belonged to 49 (92.5%), 15 (28.3%), and 11 (20.7%) A/S classes, respectively. Though all Toronto PPNG strains possessed a 24.5-Mdal conjugative plasmid, these plasmids could not be transferred by conjugation. Although some apparent microepidemics of PPNG strains were identified, most strains were isolated sporadically. CONCLUSIONS: A large number of different strains have been associated with PPNG infections in Honolulu, but there was no evidence that any strain persisted endemically during the study period. These observations have important implications for the design and assessment of community gonorrhea control strategies.

Emerging in vitro resistance to quinolones in penicillinase-producing Neisseria gonorrhoeae strains in Hawaii.

The susceptibilities of 37 penicillinase-producing strains of Neisseria gonorrhoeae (PPNG), isolated in Hawaii from December 1991 through January 1994, were determined to ciprofloxacin and ofloxacin, fluoroquinolone agents currently recommended by the Centers for Disease Control and Prevention as alternative regimens for the treatment of uncomplicated gonorrhea. Nine isolates (24.3%) exhibited decreased susceptibilities (MICs, > or = 0.06 microgram/ml) to ciprofloxacin and ofloxacin. Ciprofloxacin MICs for three isolates (8.1%) were 2.0 micrograms/ml; these isolates belonged to the auxotype/serovar class Pro/IB-7 and possessed the 3.2-MDa beta-lactamase and the 24.5-MDa conjugative plasmids. Six strains for which ciprofloxacin MICs were 0.06 to 0.125 microgram/ml belonged to a variety of gonococcal phenotypes. Strains for which ciprofloxacin MICs were 2.0 micrograms/ml were isolated from persons who had traveled to, or were sexual contacts of persons who had recently traveled to, Southeast Asia. Persons infected with these isolates had been treated with ceftriaxone (250 mg intramuscularly, single dose); therefore, none of these cases were associated with clinical failure following the use of fluoroquinolone therapy. Further studies are needed to confirm the clinical and public health significance of increased in vitro resistance to ciprofloxacin and ofloxacin in N. gonorrhoeae.

Intramolecular self-cleavage of polysialic acid.

Polysialic acid (PSA) is an unusual homopolymer of sialic acid (Sia) found on a limited number of animal glycoproteins and in the capsules of certain pathogenic bacteria. The biological properties of PSA are known to vary markedly with the length of the polymer. We confirm here that while the primary linkage unit of PSA (Sia alpha 2-8Sia) is more stable than commoner Sia linkages, PSA with > 3 Sia units is substantially more labile. A "limit digest" of PSA yields fragments of degree of polymerization (DP) = 2 and 3 and little monomeric Sia. In keeping with this, the fragmentation of PSA of DP 4 is non-random, with the internal glycosidic bond being more labile than those at the two ends. The accelerated breakdown of PSA involves an intramolecular mechanism that is not explained by lactone formation, cation effects, or specific secondary structural features. However, it is dependent upon the intactness of internal carboxyl groups, which have an anomalously high pKa. Thus, the instability of PSA appears to result from intramolecular self-cleavage of the glycosidic bonds of internal Sia units, in which the adjacent carboxyl group with a high pKa acts as a proton donor for general acid catalysis. This lability of PSA is seen under mildly acidic conditions that can be encountered in various physiological and pathological situations and thus has potential implications for neuronal adhesion, embryogenesis, and bacterial pathogenicity.

Structural, immunological, and biosynthetic studies of a sialic acid-specific O-acetylesterase from rat liver.

We have previously described a membrane-associated intralumenal sialic acid-specific 9-O-acetylesterase (LSE) from rat liver (Higa, H. H., Manzi, A., and Varki, A. (1989) J. Biol. Chem. 264, 19435-19442). Unlike a cytosolic sialate: O-acetylesterase (CSE) with similar specificity, the LSE carries N-linked oligosaccharides. A polyclonal monospecific antibody against homogenous LSE does not cross-react with the CSE. Monoclonal antibodies distinguish between the LSE and another N-glycosylated esterase that tends to partially co-purify with it. Amino-terminal sequencing of the LSE subunits indicates that it is distinct from previously described esterases and shows no homology to any other known proteins. In contrast, the esterase that partially co-purifies is similar but not identical to previously described "microsomal" esterases from rat liver. The LSE is also expressed in several hepatoma cell lines. Pulse-chase studies indicate that the two LSE subunits arise from a single precursor of approximately 65 kDa which yields a core polypeptide of apparent molecular mass approximately 53 kDa upon deglycosylation with peptide: N-glycosidase F. The protein quickly becomes partly resistant to endo-beta-N-acetylglucosaminidase H but remains sensitive to peptide: N-glycosidase F, indicating N-linked oligosaccharide processing during passage through the Golgi. After several hours, the precursor undergoes proteolysis, generating the mature heterodimeric protein of approximately 58 kDa, with subunits of approximately 38 and approximately 28 kDa. A portion of newly synthesized LSE is secreted into the medium intact, indicating that the cleavage normally takes place after diversion from the secretory pathway. These temporal changes and precursor-product distribution are reminiscent of some lysosomal acid hydrolases. In fact, immunofluorescence studies and Triton WR-1339 shift experiments suggest a lysosomal localization for this enzyme. Additional evidence for this, and the role of the LSE in O-acetylated sialic acid turnover are discussed in the accompanying paper (Butor, C., Diaz, S., and Varki, A. (1993) J. Biol. Chem. 268, 10197-10206).

Age of menopause in Japanese women: trends and recent changes.

The age of menopause was investigated in a survey conducted in 1987 in 13,996 Japanese women aged 22-86 years, the findings being as follows: (1) The percentage of post-menopausal women showed a sharp linear increase between the ages of 49 and 53 years, the figure being 42.7% at age 50. The rate of increase was slightly lower after 50 years of age. (2) Among the 6477 post-menopausal women, the age at which the peak number had undergone menopause was 50 years (17.7%), followed by 49, 52 and 48 years. The crude mean age of menopause was 49.33 years in post-menopausal subjects aged over 40. The mean age of menopause in urban and rural women was not significantly different. (3) The crude mean age of menopause, calculated by classifying those aged 50 (born in or before 1937) into groups corresponding to 5-year age intervals, was highest in the 1928-32 birth-year group, followed, in descending order, by the 1923-27, 1918-22, 1917 or earlier and 1933-37 groups in all post-menopausal and in urban subjects, while in rural subjects it was highest in the 1918-22 group. However, overall, the adjusted mean age of menopause was highest (50.2 +/- 3.24 years) in the 1928-32 birth-year group, followed by the 1923-27 and 1933-37 groups.

[Effects of catecholestrogen and catecholestrogen 2-monomethyl ether on serum lipids and lipoproteins in rats].

To clarify the mechanism of action of catecholestrogen and catecholestrogen 2-monomethylether on lipid metabolism, the effects of 2-OHE1, 2-MeoE1, 2-MeoE3 and E2-17 beta on serum total cholesterol, HDL-cholesterol, triglyceride levels, beta/alpha lipoprotein ratio, body weights and uterine weights were investigated in five serial experimental systems using normochoesterolemic and dietary hypercholesterolemic female rats those were previously oophorectomized. The results obtained were as follows: 1) In a short term hormone administration experiment using normocholesterolemic rats, 2-OHE1, 2-MeoE1, and 2-MeoE3 showed a serum triglyceride reducing effect as strong as that of E2-17 beta. 2) To integrate the results of the short term hormone administration experiment in normocholesterolemic rats and the results of short term and long term hormone administration experiments in dietary hypercholesterolemic rats, the serum cholesterol reducing activity was in the following sequences; 2-MeoE3 not equal to E2-17 beta greater than 2-MeoE1 greater than 2-OHE1. Hypocholesterolemic activity of 2-MeoE3 was almost equivalent or slightly stronger than that of E2-17 beta, and 2-MeoE1 showed approximately a half of that of E2-17 beta. 3) According to the results of the short term hormone administration experiment, and the long term hormone administration experiment in dietary hypercholesterolemic rats, the serum HDL-cholesterol increasing effect was in the following relation; E2-17 beta greater than 2-MeoE3 greater than 2-MeoE1. Dose dependency was not observed in the serum HDL-cholesterol increasing effect. 4) From the results of the short term hormone administration experiment, 2-MeoE3 had an equal or stronger activity than that of E2-17 beta in serum beta/alpha lipoprotein ratio decreasing effect. 5) In experiment 4 which 2-MeoE3 and E2-17 beta were administered singly or combined with Tamoxifen to the dietary hypercholesterolemic rats, the hypocholesterolemic effect of neither hormone was inhibited by Tamoxifen. On the other hand, the uterotrophic activity of E2-17 beta was slightly, but not significantly inhibited by Tamoxifen. 6) Although E2-17 beta, 2-MeoE1 exhibited a remarkable uterotrophic activity and a slight reducing effect on body weight, neither 2-OHE1 nor 2-MeoE3 had an effect on uterine weight or body weight. Given these results, it was strongly suggested that the effects of catecholestrogen and catecholestrogen 2-monomethyl ether on serum lipids were not mediated by the estrogen receptor system but by other mechanisms of action.

O-acetylation and de-O-acetylation of sialic acids. O-acetylation of sialic acids in the rat liver Golgi apparatus involves an acetyl intermediate and essential histidine and lysine residues--a transmembrane reaction?

Isolated intact rat liver Golgi vesicles utilize [acetyl-3H]coenzyme A to add 3H-O-acetyl esters to sialic acids of internally facing endogenous glycoproteins. During this reaction, [3H]acetate also accumulates in the vesicles, even though the vesicles are impermeant to free acetate. On the other hand, entry of intact AcCoA into the lumen of the vesicles could not be demonstrated, and permeabilization of the vesicles did not alter the reaction substantially (Diaz, S., Higa, H. H., Hayes, B. K., and Varki, A. (1989) J. Biol. Chem. 264, 19416-19426). When vesicles prelabeled with [acetyl-3H] coenzyme A are permeabilized with saponin, we can demonstrate a [3H]acetyl intermediate in the membrane that can transfer label to the 7- and 9-positions of exogenously added free N-acetylneuraminic acid but not to glucuronic acid or CMP-N-acetylneuraminic acid. This labeled acetyl intermediate represents a significant portion of the radioactivity incorporated into the membranes during the initial incubation and cannot be accounted for by nonspecifically "trapped" acetyl-CoA in the permeabilized vesicles. There was no evidence for involvement of acetylcarnitine or acetyl phosphate as an intermediate. The overall acetylation reaction appears to involve two steps. The first step (utilization of exogenous acetyl-CoA to form the acetyl intermediate) is inhibited by coenzyme A-SH (apparent Ki = 24-29 microM), whereas the second (transfer from the acetyl intermediate to sialic acid) is not affected by millimolar concentrations of the nucleotide. Studies with amino acid-modifying reagents indicate that 1 or more histidine residues are involved in the first step of the acetylation reaction. Diethylpyrocarbonate (which can react with both nonsubstituted and singly acetylated histidine residues) also blocks the second reaction, indicating that the acetyl intermediate on both sides of the membrane involves histidine residue(s). Taken together with data presented in the preceding paper, these results indicate that the acetylation of sialic acids in Golgi vesicles may occur by a transmembrane reaction, similar to that described for the acetylation of glucosamine in lysosomes (Bame, K. J., and Rome, L. H. (1985) J. Biol. Chem. 260, 11293-11299). However, several features of this Golgi reaction distinguish it from the lysosomal one, including the nature and kinetics of the reaction and the additional involvement of an essential lysine residue. The accumulation of free acetate in the lumen of the vesicles during the reaction may occur by abortive acetylation (viz. transfer of label from the acetyl intermediate to water). It is not clear if this is an artifact that occurs only in the in vitro reaction.

O-acetylation and de-O-acetylation of sialic acids. 7- and 9-o-acetylation of alpha 2,6-linked sialic acids on endogenous N-linked glycans in rat liver Golgi vesicles.

We have previously shown that radioactivity from [acetyl-3H]AcCoA is concentrated into isolated intact rat liver Golgi vesicles. The incorporated radioactivity occurred in acid-soluble and acid-insoluble components, and the acid-insoluble fraction included O-acetylated sialic acids (Varki, A., and Diaz, S. (1985) J. Biol. Chem. 260, 6600-6608). Nearly all of the protein-associated radioactivity was found to be in sialic acids alpha 2-6-linked to N-linked oligosaccharides on endogenous glycoproteins. Incubation of the vesicles with CMP-[3H]sialic acid resulted in labeling of a very similar group of glycoproteins. The 3H-O-acetyl groups were found at both the 7- and the 9-positions of N-acetylneuraminic acid residues at the end of the labeling reaction. Although 7-O-acetyl groups can undergo migration to the 9-position under physiological conditions, kinetic studies using O-acetyl-14C-labeled internal and O-acetyl-3H-labeled external standards indicate that during the labeling, release, and purification, negligible migration occurred. Studies with mild periodate oxidation provided further confirmation that O-acetyl esters are added directly to both the 7- and the 9-positions of the sialic acids in this system. The acid-soluble, low molecular weight component is released from the vesicles by increasing concentrations of saponin, and its exit parallels that of CMP-[14C]sialic acid taken up during the incubation. The vesicles themselves are impermeant to free acetate. However, even after short incubations, this saponin-releasable radioactivity was almost exclusively in [3H] acetate and not in [3H]acetyl-CoA. The apparent Km for accumulation of the [3H]acetate is almost identical with that for the generation of the acid-insoluble O-acetylated sialic acids. Most of this accumulation of free acetate is also blocked by coenzyme A-SH. Only a small portion arises from the action of an endogenous esterase on the 3H-O-acetylated sialic acids. Taken together, the results indicate that accumulation of free [3H]acetate occurs within the lumen of the vesicles in parallel with O-acetylation of sialic acids and is probably a product of abortive acetylation. It is not known if this reaction occurs in vivo. Permeabilization of Golgi vesicles to low molecular weight molecules with saponin does not alter the rate of acetylation substantially. Furthermore, double label studies suggest that the intact acetyl-CoA molecule does not gain access to the lumen of the vesicles. These results indicate that the acetylation reaction may have a different mechanism from previously described Golgi glycosylation reactions, wherein specific transporters concentrate sugar nucleotides for use by luminally oriented transferases.

O-acetylation and de-O-acetylation of sialic acids. Purification, characterization, and properties of a glycosylated rat liver esterase specific for 9-O-acetylated sialic acids.

We have previously described the preparation and use of 9-O-[acetyl-3H]acetyl-N-acetylneuraminic acid to identify sialic acid O-acetylesterases in tissues and cells (Higa, H. H., Diaz, S., and Varki, A. (1987) Biochem. Biophys. Res. Commun. 144, 1099-1108). All tissues of the adult rat showed these activities, with the exception of plasma. Rat liver contained two major sialic acid esterases: a cytosolic nonglycosylated enzyme and a membrane-associated glycosylated enzyme. The two enzymes were found in similar proportions and specific activities in a buffer extract of rat liver acetone powder. By using the latter as a source, the two enzymes were separated, and the glycosylated enzyme was purified to apparent homogeneity by multiple steps, including ConA-Sepharose affinity chromatography and Procion Red-agarose chromatography (yield, 13%; fold purification, approximately 3000). The homogeneous enzyme is a 61.5-kDa disulfide-linked heterodimeric protein, whose serine active site can be labeled with [3H]diisopropyl fluorophosphate. Upon reduction, two subunits of 36 kDa and 30 kDa are generated, and the 30-kDa subunit carries the [3H]diisopropyl fluorophosphate label. The protein has N-linked oligosaccharides that are cleaved by Peptide N-glycosidase F. These chains are cleaved to a much lesser extent by endo-beta-N-acetylglycosaminidase H, indicating that they are mainly complex-type glycans. The enzyme activity has a broad pH optimum range between 6 and 7.5, has no divalent cation requirements, is unaffected by reduction, and is inhibited by the serine active site inhibitors, diisopropyl fluorophosphate (DFP) and diethyl-p-nitrophenyl phosphate (Paraoxon). Kinetic studies with various substrates show that the enzyme is specific for sialic acids and selectively cleaves acetyl groups in the 9-position. It shows little activity against a variety of other natural compounds bearing O-acetyl esters. It appears to deacetylate di-O-acetyl- and tri-O-acetyl-N-acetylneuraminic acids by first cleaving the O-acetyl ester at the 9-position. The 7- and 8-O-acetyl esters then undergo spontaneous migration to the 9-position, where they can be cleaved, resulting in the production of N-acetylneuraminic acid. In view of its interesting substrate specificity, complex N-linked glycan structure, and neutral pH optimum, it is suggested that this enzyme is involved in the regulation of O-acetylation in membrane-bound sialic acids.

Acetyl-coenzyme A:polysialic acid O-acetyltransferase from K1-positive Escherichia coli. The enzyme responsible for the O-acetyl plus phenotype and for O-acetyl form variation.

The capsular polysaccharide of Escherichia coli K1 is a linear polymer of N-acetylneuraminic acid in alpha-2,8 linkage. Certain substrains of E. coli K1 (designated OAc+) modify the polysaccharide by O-acetylation of the sialic acids. We demonstrate here an acetyl-coenzyme A: polysialosyl O-acetyltransferase activity that is found only in E. coli K1 OAc+ substrains. When form variation between the O-acetyl-positive and -negative states occurred in strain D698:K1, the fluctuations were accompanied by appropriate changes in the expression of enzyme activity. Thus, expression of this enzyme can account for the OAc+ phenotype and for the form variation between OAc+ and OAc-. The enzyme was solubilized in nonionic detergent and freed of endogenous acceptor activity by DEAE-cellulose chromatography, and its general properties were determined. Analysis of the reaction product showed a highly preferential acetylation reaction that was confined to polysialosyl units of greater than 14 residues. Acetyl groups were shown to be transferred to both the 7- and the 9-positions of the sialic acid residues. The partially purified enzyme was stable even after prolonged incubation at 57 degrees C. In contrast, any further purification resulted in loss of activity, even at 4 degrees C. Treatment of the stable enzyme with a polysialic acid-specific endoneuraminidase caused a similar loss of enzyme stability. This effect of the endoneuraminidase could be protected against by the addition of exogenous polysialic acid. This indicates that the partially purified enzyme contains traces of endogenous polysialic acid substrate that are required for the stability of the enzyme. Finally, the enzyme can O-acetylate the polysialic acid chains on the eucaryotic protein neural cell adhesion molecule, suggesting that enzymatic recognition of the substrate requires only the polysialic acid sequence.